Metastatic melanoma is the deadliest form of skin cancer and one of the most aggressive cancer types, capable of rapid spreading out of the primary site. This proposal is aimed at analyzing the combined effect of changes in the genetic and signaling states of melanoma cells and the cell micro-environment on the degree of cell aggressiveness and putative metastatic potential. The main hypothesis underlying our analysis is that aggressiveness of melanoma cells is determined by a combination of different factors, with a major contribution from the state of cell microenvironment, including the structure of the ECM and the presence of soluble chemical factors. Alteration in the cell micro-environment, such as alignment of ECM fibers, can permit an otherwise non-metastatic cell to become more aggressive, whereas the aggressiveness of ostensibly metastatic cell can be moderated by environmental changes and targeted intervention in regulation of signaling cascades. We also hypothesize that cells can actively interact with the micro-environment not only by secreting or degrading ECM components, but by actively exercising forces on and thus aligning ECM fibers in the direction of a nearest anchor point, including the vasculature. This can in turn generate more anisotropic forces allowing cells to deform ECM even more and ultimately take advantage of the re-aligned ECM structure to migrate towards blood vessels. We will test these hypotheses in close collaboration with ICBP@MIT, first taking advantage of the new methodologies using shRNA libraries to perturb genetic targets (Aim 1) along with a broad range of environmental perturbations and then use the state of the art statistical analysis tools available at ICBP@MIT to reduce complexity of resulting datasets, linking multiple environmental and genetic perturbation to aggressive cell behavior (Aim 2), We will use several novel assays in the process, increasing manageability of data acquisition and processing, as well as enhancing the biomimetic nature of experimentation. Finally, we will test the predictions arising from the models developed in Aim 2 through a series of tests, ranging from cell culture to tissue construct to model tissue levels (Aim 3). We anticipate that the proposed research will reveal new, previously unanticipated interplay between environmental and genetic factors in control of metastatic events in melanoma and potentially suggest new ways of battling this deadly disease. The results might also have relevance to other metastatic cancers. PUBLIC HEALTH RELEVANCE: Early detection is further hampered by the lack of biomarker-based detection tests. One of the critical reasons for this situation is the lack of understanding of the mechanisms underlying the metastatic switch in melanoma and the mechanisms allowing metastatic cells to successfully and quickly navigate all the hurdles associated with the metastatic spread, including effective migration to and intravasation into the blood vessels. This project is aimed at filling this critical gap.